689. The Content of the More Immature Hematopoietic Stem Cells (HSCs) Is Dependent from the Underlying Genetic Diseases: Consequence on Transduction Efficiencies

Abstract

Ex vivo gene transfer into hematopoietic CD34+ cells provides therapeutic benefits in primary immunodeficiencies. However, the CD34+ cell population is heterogeneous and is mainly composed of progenitors (CD34+lin-CD38+) and multilineage progenitors (CD34+lin-CD38-). This latter population can be divided in 3 subtypes regarding to the CD45RA and CD90 expression. CD45RA+ define cells with lymphoid potential, CD45RA-CD90- defined cells with myeloid potential and CD45RA-CD90+ define cells with long term repopulating capability (HSCs) which constitutes the relevant target for gene correction. We found that in bone marrow (BM) CD34+linneg, addition of CD133 helps to delimitate CD38neg population and we have analysed HSC, MLP and MPP population in BM from SCID-X1 (n=4), Wiskott Aldrich Syndrome (WAS) (n=2), Artemis (n=2), Chronic Granulomatous Disease (CGD) patients (n=3), sickle cell disease (SCD) (n=3) and age matched healthy controls (n=6).As expected, mean percentage of HSC represent 1% of CD34+ in healthy donors and similar results are obtained in CD34+ from SCID-X1 (1.76% ± 0.72), Artemis (1.98% ± 0.15), and SCD (2.16% ± 0.6) patients. On the contrary HSC percentage are lower in CD34+ from WAS (0.35% ± 0.15) and nearly undetectable in CGD patients. Moreover in multilineage progenitors, proportion of HSC, MLP and MPP varies from one pathology to another as shown in table1table1.Table 1Mean percentage of HSC, MLP and MPP in the linnegCD133+CD38neg CD34+ cellsHSCsMLPMPPHealthy Donor42%33%24%SCIDX137%45%28%WAS35%35%30%Artemis30%48%20%SCD36%19%44%Our aim was then to determine the ability of vectors used for gene therapy to transduce these different progenitors. We first analysed transduction of bulk CD34+ and HSCs by SIN retroviral vector for IL2RG in 2 SCIDX-1 patients enrolled in the clinical trial. After transduction, the 3 cell types were purified by flow cytometry and transduction determined in CFU after 14 days of culture in methyl cellulose in order to avoid detection of non-integrated vector. We show that in both patients, transduction efficiency was lower in HSCs than in bulk CD34+ cells. We were not able to recover enough CFU to determine VCN from MPP fraction and as expected, no CFU were obtained from MLP fraction. These results will be extended to transduction of CD34+ from WAS and SCD patients and efforts will be concentrated on transduction protocols in order to improve transduction of HSCs. Nevertheless low HSCs contents observed in CD34+ from CGD patients also represents a major difficulty to obtain long term reconstitution after gene therapy.